Construction of a SV40 Virus Large J Antigen Eukaryocyte Vector and Its Targeted Expression / 中华医院感染学杂志

ABSTRACT

OBJECTIVE To design and construct eukaryocyte expression vector of SV40 virus large T antigen and induce its targeted expression in eukaryocyte.METHODS SV40 large T gene which excised intron was cloned by SOE(splicing by overlapping extension) and digested with restricted enzymes EcoR Ⅰ and BamH Ⅰ.By the same methods,we got the digested product of pEGFP-N1.After that,the two fragments were ligated to form SV40(TEGFP) by Ligation Kit,and sequenced by TaKaRa ABI Prism Terminator Cycle Sequence Kit.The reconstructed vector was transfected into primary cultured human fibroblast using a Lipofectin transfection method.At 48 h(after) transfection,the expression of SV40T was detected with PCR and RT-PCR using specific primer of T gene.(RESULTS) The restricted enzymes digested and sequencing results showed that SV40 large T gene had cloned into pEGFP-N1 vector successfully.The genome DNA and total RNA were isolated from the positive cells.With these samples,the specific 288 bp fragment was amplified using PCR and RT-PCR.CONCLUSIONS The recombinant plasmid SV40TEGFP will be a stable and valuable molecular tool for human eukaryocyte study.